The most notable characteristic feature of onion is that a large amount of a lachrymatory factor (hereinafter referred to as “LP”) is generated when it is pulverized or cut. Therefore, the generation of LF is a serious problem not only in cooking in ordinary kitchens but also in factories for producing dry onion. Under these conditions, various investigations were made on the chemical structure of LF and the generation process thereof. It was reported that LF is essentially thiopropanal S-oxide (Wilkins, W. F., ph. D. thesis, Cornell University, Ithaca, N.Y., 1961), that S-1-propenyl-cystein sulfoxide (hereinafter referred to as “PeCSO”) which is a sulfur-containing compound contained in onion is decomposed by alliinase (Virtanen, A. I. et al. Suom. Kemistil. B, 34, 72, 1961) and that LF is generated through 1-propenylsulfenic acid which is a decomposition product of PeCSO with alliinase (Block, e. et al., J. Am. Chem. Soc., 11, 2200, 1979).
Because it was considered that LF is formed by the decomposition of PeCSO with alliinase in the prior art, there was proposed a method for producing onion generating a reduced amount of LF, which comprises the production of onion having a reduced PeCSO content or the production of onion having a reduced alliinase activity.
Under the circumstances, investigations were made for the purpose of varying the amount of PeCSO accumulated in onion by varying the cultivating conditions. For example, it was reported that when onion is cultivated under a low sulfur content condition, LF content is reduced (Randle, W. M. et al., J. Agr. Food Chem. 42, 2085, 1994) and the relative amount of PeCSO to the substrate of alliinase is also reduced (Randle, W. M. et al., J. Amer. Soc. Hort. Sci. 120, 1075, 1995). It was also reported that when onion is cultivated in the presence of selenium, the product has a reduced PeCSO content (Kopsell, D. E. et al., J. Amer. Soc Hort. Sci. 124, 307, 1999) and PeCSO content of the obtained onion is increased during the storage thereof (Kopsell, D. E. et al., J. Amer. Soc Hort. Sci. 124, 177, 1999) and that the larger the amount of ammonium nitrate used as a fertilizer, the lower PeCSO content (Randle, W. M. et al., J. Amer. Soc Hort. Sci. 125, 254, 2000).
However, the onion cultivated in the presence of a reduced amount of PeCSO has a problem that the smell is weakened (p. 41-52. In: S. J. Risch and C. Ho(eds.). Spices: Flavor chemistry and antioxidant properties. Amer. Chem. Soc., Wash., D.C.) and the relative amount of PeCSO to the substrate of alliinase is changed to change of the quality of the smell per se. Thus, only the change in the cultivation conditions is not a fundamental solution for the following reason: 1-Propenylsulfenic acid produced by the decomposition of PeCSO with alliinase is converted into not only LF but also thiosulfinate compounds which are the source of the smelling ingredients.
The applicant found a lachrymatory factor-producing enzyme capable of converting 1-propenylsulfenic acid into LF and applied it for patent (Japanese Patent Unexamined Published Application (hereinafter referred to as “J. P. KOKAI”) No. Hei 10-295373). The applicant further elucidated an isozyme of this lachrymatory factor-producing enzyme, amino acid sequence thereof and DNA encoding the isozyme, and applied them for patent (International Patent Application PCT/JP01/07465).
Supposedly, if the expression of the lachrymatory factor-producing enzyme can be repressed and the activity thereof is inhibited by using the lachrymatory factor-producing enzyme elucidated in the above-described patent application, LF is not produced from 1-propenylsulfenic acid but the thiosulfinate compounds which give off the smell can be produced in an amount not less than that in the prior art irrespectively of the effect of the enzyme. In addition, according to genetic information of a gene encoding the lachrymatory factor-producing enzyme, the genetic recombination, induction of variation, and mating can be effectively conducted. Additionally, a technique of producing vegetables such as onion in which the lachrymatory factor is not easily formed by the pulverization or cutting thereof can be developed.
The main point of the present invention is to obtain vegetables having repressed expression of the lachrymatory factor-producing enzyme by a genetic engineering technique for efficiently producing intended vegetables in a short period according to the sequence information of the gene encoding the lachrymatory factor-producing enzyme of the prior application.
Namely, the object of the present invention is to provide DNA and RNA designed on the basis of the sequence of a gene of an enzyme for forming the lachrymatory factor from a precursor of this factor for the purpose of repressing the expression, and also a vector required for introducing the expression-repressing DNA of the gene of the lachrymatory factor-producing enzyme into a vegetable. Another object of the present invention is to provide a method for repressing the expression of the gene of the lachrymatory factor-producing enzyme by using them and also a vegetable in which the expression of the gene of the lachrymatory factor-producing enzyme is repressed. The present invention has great advantages that because the formation of the lachrymatory factor can be essentially repressed, the onion is not influenced by other external factors and also that because no influence is exerted on the amount of the precursor of the lachrymatory factor, the quality of onion is not lowered. Another advantage of the present invention is that the expression of the gene can be repressed in a period shorter than that in ordinary techniques of breeding vegetables which are free from the genetic engineering.